Cell culture technology has become a well-established technique that is very successful in vitro under ideal laboratory conditions. With cell specific culture media, growth factors, nutrients, and temperature, almost all human cell lines have been successfully grown in the laboratory.
Scaffold technology has made multilayer tissue engineering possible as well, with multi-cell structures successfully grown in the laboratory. Despite these successes, major roadblocks still exist in translational research. As a consequence, the only organ successfully engineered to date is a urinary bladder (Atala et al., Lancet, vol. 367, p. 1241-6, 2006).
A short list of persistent problems in this area include: 1) lack of a well developed vascular supply for organogenesis; 2) tissue resorption; 3) loss of cell function; and 4) untoward side effects.
As relates specifically to item 1), almost all tissue engineering is done with non-vascularized scaffolds. Although neovascularization with capillaries occurs very reliably in scaffolds about 1 mm in thickness, most human organs are much larger than this. As a consequence, tissue engineering on scaffolds is limited in size by the lack of arterial and venous structures which do not grow as well as capillaries. In summary, vascular supply limits organ size in scaffold based tissue engineering.
As relates specifically to item 2), tissue resorption often occurs when non-vascularized grafts are transferred in human autograft transplantation. All human autografts undergo this resorption even in the absence of infection, antigen-antibody mismatch, or lack of nutrition. A list of the tissues which have been autografted with well documented resorption over time include a) fat grafts—fat grafts larger than a few mm in diameter are well documented of undergoing resorption over time. Except for small volume fat grafting transferred into multiple well vascularized tunnels, most fat grafts undergo partial resorption. The most disconcerting aspect is that the resorption rate varies widely from 20% to 90%! This makes it difficult to compensate for resorption by overgrafting with larger volumes; b) bone grafts—small, thin non-vascularized bone grafts have an excellent track record of success in human autotransplantation. Grafts larger than 6 cm in length usually do not neovascularize as well and undergo major resorption. For this reason, most large defects are reconstructed with vascularized osteocutaneous flaps. These microvascular free flaps allow for the transfer of large bone grafts up to 24 cm in length. This difference in bone graft resorption between vascularized and nonvascularized bone grafts illustrates why this issue is pivotal in the development of tissue engineering on the multi-cm scale; and c) cartilage grafts—since cartilage is one of the few human tissues that has no endogenous blood supply and is nourished only by diffusion from the surrounding perichodrium, there was great hope that cartilage grafts would be good model for tissue engineering that would not be as dependent on vascularization for success. The full-size human ear cartilage that was successfully engineered on the back of a mouse was hailed as a major breakthrough. Sadly, this ear cartilage also underwent resorption as the ear framework scaffolding resorbed. To this date, cartilage autografts are well documented as having higher resorption rates than bone grafts in nasal surgery, craniofacial surgery, joint surgery, and ear reconstruction. Small, thin grafts do well in children. Large, thick grafts in adults fare poorly.
As relates specifically to item 3), loss of cell function also occurs in autografts involving well differentiated cells such as endocrine cells, paracrine cells, epithelium with cilia, or exocrine cells. In these cases, cell function may be lost after grafting despite cell survival. Although the exact cause for this loss of function is not clearly known, lack of a complete vascular bed (arteries, capillaries and veins) in these models may be a contributing factor. Some examples of grafts that lose their function include: a) hepatocyte grafts—in many models, hepatocyte grafting has been successful but hepatic function is lost in transfer for a significant fraction of the cells; b) B-islet cell grafts—insulin producing cells have been successfully grown in the laboratory and transplanted into animal and human subjects. Despite this success, there is a major loss in insulin production by these cells; and c) dopaminergic cell grafts—the focus of research today for the treatment of Parkinson's disease has been directed towards dopaminergic cell replacement therapy. Although these highly specialized cells have been successfully cultured, transplanted into the human brain, cell function is gradually lost.
As relates specifically to item 4), untoward side effects also occur in human autografting when cell transplantation occurs in scaffolds. A few of the well documented sequelae that have stymied translational research include: a) heterotopic ossification—when osteoprogenitor cells are transferred on scaffold systems with the necessary growth factors (bone morphogenic proteins) in children, heterotopic ossification of the surrounding muscles often result in pain, muscle function loss, and disability. Heterotopic ossification is not reversible when the scaffold is removed or when the BMPs are gone. This has been major road block in pediatric translational research despite successful osteoblast cell survival with good osteoblast function; b) scar tissue formation—due to limitations on graft size, most scaffold based tissue engineered grafts develop scar tissue around the graft. This may be due in part to normal collagenesis that occurs in wound healing, but may also be due to ischemia which is a well known trigger for scar tissue formation. Foreign body reaction to the scaffold itself may also be a cause of this scar tissue formation. Regardless of the cause, scar tissue is a major factor in disrupting normal organ function. This is considered to be an important problem in hepatic cirrhosis, glomerulonephritis, interstitial lung disease, and tenosynovitis; and c) calcifications—when fat is transferred by autologous nonvascularized grafting, by pedicled flaps, or by free microvascular flap transfer, fat necrosis is a minor occurrence with major consequences. Even if only a small percentage of the fat cells undergo cell death, these dead cells undergo saponification releasing abundant long chain fatty acids from the disrupted plasma membrane. Precipitation of these long chain fatty acids with calcium results in palpable masses that appear on mammography to be microcalcifications. This artifact makes cancer surveillance difficult with mammography. As a consequence, fat grafting for breast augmentation may make future cancer detection difficult. For this reason, autologous fat grafting for cosmetic breast augmentation has been discouraged by the FDA, radiological societies, and the plastic surgery community. Fat necrosis also causes concerns when it occurs in the reconstructed breast. In this scenario, both patients and their oncologists worry that the palpable fat necrosis may be recurrent cancer. This often necessitates a biopsy to rule out this possibility.
There has been extensive research by others to develop biocompatible composites/scaffolds, etc. For example, Burg described a biocompatible composite with viscous fluid for injection into defects (US Pat. appl. 20020022883 A1 by Burg). Of course, this concept would not work for organogenesis. Sahatjian et al. proposed a three dimensional cell scaffold either as a sheet or a tube configured into various shapes (US Pat. appl. 20040126405 by Sahatjian et al.). The Harvard University group led by Vacanti proposed placing dissociated cells into a biodegradable matrix to be implanted with a tissue expander device into the breast (U.S. Pat. No. 5,716,404 by Vacanti et al.). However, cells would perish without new blood vessels, and this idea did not materialize into practical use since its issue in 1998. Vacanti et al. also reported the idea of implanting sheets of cell-matrix structure adjacent to mesentery, omentum, or peritoneum tissue (see U.S. Pat. No. 5,804,178, U.S. Pat. No. 5,770,193, and U.S. Pat. No. 5,759,830).
Yelick et al. successfully constructed a biodegradable polymer scaffold molded in the shape of a tooth and placed in onto the omentum of rats (US Pat. appl. 20020119180 A1 by Yelick et al.). A later application describes a method to achieve high density seeding of polymer scaffold with organoid units (US Pat. appl. 20030129751 by Grikscheit et al.). The disclosed scaffolds are collagen-coated 1 cm long 0.5 mm woven polyglycolic acid tubes with a diameter of 0.5 cm, that are sutured to the rat's omentum to make new colonic tissue (Grikscheit et al., Annals of Surgery, vol. 238, p. 35-41, 2003).
The omentum has been used by various investigators as a source of vasculature for tissue engineering purposes: porcine tooth (Sumita et al., Biomaterials, vol. 27, p. 3238-48, 2006), dog small intestine (Hori et al., International Journal of Artificial Organs, vol. 24, p. 50-4, 2001), dog tracheal defects (Kim et al., Journal of Thoracic and Cardiovascular Surgery, vol. 128, p. 124-9, 2004), canine oral epithelial cells and rib chondrocytes (Suh et al., ASAIO Journal, vol. 50, p. 464-7, 2004), and porcine bladder urothelial cells (Moriya et al., Journal of Urology, vol. 170, p. 2480-5, 2003). The Vacanti group also used the mesentery and interscapular fat pad to grow hepatocytes, intestinal cells and pancreatic islet cells in mice and rats (Vacanti et al., Journal of Pediatric Surgery, vol. 23, p. 3-9, 1988). Recently, a successful human clinical trial has been reported (Atala et al., Lancet, vol. 367, p. 1241-6, 2006; US Pat. appl. 20070059293 A1 by Atala). Autologous bladder cells were seeded on a biodegradable bladder-shaped scaffold made of collagen and polyglycolic acid, which was then implanted covered with omentum into the patients with myelomeningocele. In all of the above studies, the omentum was used as a single layer attached to one side of a flat scaffold, or wrapped around a three-dimensional scaffold.
Due to the problems listed above and despite the extensive research described above, tissue engineering on non-vascularized scaffolds has reached a major obstacle in developing organ structures greater than a few mm in size. Since most organs are larger than this, a better scaffold is needed. The present inventors set forth to address this important unmet need.